Neurogenic mechanisms in control of the rabbit iris sphincter muscle.

The iris sphincter muscle is supplied with cholinergic, adrenergic and substance P-containing nerve fibers, all with a possible role in the control of pupil size. The functional significance of the various nervous components in the rabbit iris sphincter muscle was examined in vitro and in vivo. The contractile response to electrical stimulation is composed of several contractions, occurring along different time scales. Single pulse stimulation produced an atropine-sensitive twitch. Pulse train stimulation revealed two successive atropine-sensitive twitches followed by a slow, long-lasting contraction, sensitive to Spantide, an antagonist of tachykinins such as substance P. A guanethidine- and phentolamine-sensitive contractile response to pulse train stimulation could be demonstrated in the presence of both atropine and Spantide. Only the Spantide-sensitive response could be completely exhausted by prolonged electrical stimulation. In vivo, neither the adrenergic nor the substance P-containing nerve fibres appeared to contribute to the miotic response to light since Spantide and guanethidine were without effect. This response was inhibited by atropine only.

Sympathectomy enhances the substance P-mediated breakdown of the blood-aqueous barrier in response to infrared irradiation of the rabbit iris.

Rabbits were subjected to infrared irradiation of the iris 1 month after unilateral cervical sympathectomy. The resulting breakdown of the blood-aqueous barrier was greatly enhanced on the sympathectomized side. In contrast, the response to intravitreally injected substance P (SP) was the same in both eyes. The enhancement of the response to IR irradiation could be abolished by pretreatment with an SP antagonist, (D-Pro2, D-Trp7,9)-SP.

Pupillary supersensitivity to substance P following prolonged treatment with tetrodotoxin or substance P antagonists.

Pilocarpine contracts the sphincter pupillae muscle via an effect on muscarinic receptors and phenylephrine contracts the dilator pupillae muscle via an effect on alpha-adrenergic receptors. These effects are thought to mimic the action of the parasympathetic and sympathetic nervous systems, respectively. Intracellular injection of substance P (SP) produces an atropine-resistant constriction of the pupil. This response is thought to mimic the effect of local sensory reflexes on the sphincter pupillae muscle, involving SP-containing trigeminal nerve endings. Repeated intraocular injections of tetrodotoxin, a general blocker of nervous conduction, over a period of 3 weeks produced supersensitivity to pilocarpine, phenylephrine and SP in the rabbit iris. These findings support the view that, like acetylcholine and noradrenaline, SP or an SP-like compound acts as a neurotransmitter in the iris. Also, long-term topical application of an SP antagonist, (D-Pro2,D-Trp7,9)-SP or (Arg5,D-Trp7,9)-SP5-11, to the rabbit eye produced supersensitivity to SP but not to pilocarpine, thus supporting the view that the SP antagonists interact specifically with the SP receptors. The isolated rabbit iris sphincter muscle responds to electrical stimulation with a cholinergic twitch followed by a slow non-cholinergic contraction that can be blocked by antagonists to SP. Analysis of the motor activity of the iris sphincter muscle after long-term topical treatment of the eye with an SP antagonist followed by an interval of 2 days after termination of treatment revealed a greatly enhanced non-cholinergic contraction compared with the cholinergic twitch, a finding that seems to be consistent with the idea that supersensitivity to SP had developed.

Pupillary constriction by bradykinin and capsaicin: mode of action.

The application of bradykinin or capsaicin to the rabbit eye evoked strong miosis. The effect could be prevented by pretreatment of the eye with tetrodotoxin (TTX) or a substance P (SP) antagonist. However, the miotic response could be elicited despite TTX or the SP antagonist if the dose of capsaicin or bradykinin was increased. Bradykinin and capsaicin contracted the isolated rabbit sphincter pupillae muscle. The contraction produced by bradykinin and capsaicin was unaffected by TTX but reduced by specific SP antagonists. This indicates that bradykinin and capsaicin exert their effects on the isolated sphincter muscle through the release of SP but independent of neuronal conduction. In vivo, the situation seems to be different. The finding that TTX is capable of blocking the miotic response to moderate doses of bradykinin and capsaicin suggests that the effect on the eye under these circumstances is dependent upon a normal impulse traffic.

Is substance P necessary for corneal nociception?

Substance P (SP)-containing nerve fibers are few in the rabbit cornea, which is richly supplied with acetylcholinesterase-positive nerve fibers, presumably sensory in nature. Treatment with capsaicin given by retrobulbar injection 48 h prior to sacrifice caused SP to disappear from the SP nerves in the cornea without affecting the acetylcholinesterase-positive nerve fibers. The corneal sensitivity to a tactile stimulus (wetted cotton swabs) and to a chemical stimulus (local application of capsaicin) and the disruption of the blood-aqueous barrier after local injury (infrared irradiation of the iris) were tested after pretreatment with capsaicin (retrobulbar injection) the SP antagonist [D-Pro2,D-Trp7,9]SP (single topical application or long-term application twice daily for 2 months), the local anaesthetic oxibuprocaine (topical application), or the neuronal blocker tetrodotoxin (intravitreal injection). All these treatments abolished or reduced the disruption of the blood-aqueous barrier after local injury but only oxibuprocaine and tetrodotoxin abolished the corneal sensitivity to tactile and chemical stimuli. It is suggested that SP nerve fibers constitute a minor proportion of the sensory nerve supply to the cornea, that the neurogenic mechanisms involved in the response to ocular injury differ from those involved in corneal nociception, and that uveal SP is involved in the response to ocular trauma and that corneal SP is probably not necessary for corneal nociception.

Long-term administration of a substance P antagonist, (D-Pro2, D-Trp7,9)-SP, abolishes the response to ocular trauma.

The long-term effect of ocular administration of a substance P (SP) antagonist, (D-Pro2, D-Trp7,9)-SP, was studied in the rabbit. After 2-3 months of topical administration of the antagonist twice daily, a mild trauma was applied in the form of infrared irradiation of the iris. The control eye responded with disruption of the blood-aqueous barrier, the eye treated with the antagonist did not. Two days after termination of treatment, the response to ocular injury was still reduced. Another 2 days later, ocular injury evoked a normal response, which shows that the protection was reversible. No adverse reaction to the SP antagonist was noted.

Ocular responses evoked by capsaicin and prostaglandin E2 are inhibited by a substance P antagonist.

Injection of capsaicin or prostaglandin E2 into the vitreous chamber of the rabbit eye resulted in miosis and breakdown of the blood-aqueous barrier, manifested in aqueous flare. Pretreatment with the neuronal blocker tetrodotoxin or the substance P antagonist (D-Pro, D-Trp)-SP greatly reduced the ocular responses to capsaicin and prostaglandin E2. The results suggest a role for neuronal substance P in the ocular response to injury.

Bradykinin contracts the pupillary sphincter and evokes ocular inflammation through release of neuronal substance P.

Bradykinin contracts the isolated rabbit sphincter pupillae muscle. The contraction produced by 10(-8) M bradykinin was resistant to atropine but not to tetrodotoxin, suggesting a non-cholinergic nervous mechanism. The contraction was blocked by specific substance P (SP) antagonists, suggesting the involvement of SP. The SP antagonists tested were [D-Pro2,D-Trp7,9]SP-(1-11) and [Arg5,D-Trp7,9]SP-(5-11). The bradykinin-induced contraction exhibited marked tachyphylaxis in contrast to that induced by SP. It appears that the tachyphylaxis reflects the depletion of a bradykinin-sensitive neuronal pool of SP. Injection of bradykinin into the vitreous chamber of the rabbit eye caused miosis and disruption of the blood-aqueous barrier (manifested as aqueous flare). A second administration of bradykinin a few hours after the first injection evoked a reduced response; the response to SP upon repeated administration was unchanged. Atropine was without effect on the response to bradykinin whereas tetrodotoxin and the SP antagonists reduced the response. The results suggest that bradykinin causes miosis and aqueous flare at least partly through local release of neuronal SP.

Capsaicin pretreatment prevents disruption of the blood-aqueous barrier in the rabbit eye.

Capsaicin, the irritating agent of red pepper, produces ocular inflammation through a neurogenic mechanism. The present study is concerned with the long-term effects of capsaicin pretreatment on the capacity of the eye to respond to different inflammatory stimuli. Following retrobulbar injection of capsaicin to rabbits the aqueous flare response induced by subsequent infrared irradiation (IR) of the iris, subcutaneously administered alpha-melanocyte-stimulating hormone (alpha-MSH) and exogenously administered prostaglandin E2 (PGE2) was reduced greatly. In the case of IR and alpha-MSH the reduced responsiveness was manifest for several weeks after capsaicin pretreatment, involving first the capsaicin-treated eye, but later also the contralateral control eye. After 2-3 months the aqueous flare response was normal in both eyes. In the case of PGE2 the responsiveness was reduced for a shorter time; after 3 weeks the response was normal in both eyes. The results indicate that all three stimuli tested are at least partly dependent upon an intact sensory innervation to disrupt the blood-aqueous barrier, but that the mechanism of action of PGE2 is different from that of IR and alpha-MSH.